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Original Article

Effects of chronic occupational exposure to anaesthetic gases on the rate of neutrophil apoptosis among anaesthetists

Tyther, R.*; Halligan, M.*; Wang, J.; Redmond, H. P.; Shorten, G.*

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European Journal of Anaesthesiology: August 2002 - Volume 19 - Issue 8 - p 604-608



At present, evidence about the potential adverse effects of anaesthetic agents in the working environment is inconclusive. However, it is likely that the presence of anaesthetic gases in the atmosphere is undesirable. In addition to being implicated in carcinogenic and teratogenic effects [1-3], Peric and colleagues [4] demonstrated that environmental exposure to anaesthetic gases causes immunological disturbances in anaesthetic personnel. Anaesthetic staff chronically exposed to high occupational concentrations of halothane and nitrous oxide were tested for haematological function during a period of work and after a 3 week holiday. After withdrawal from exposure, the numbers of circulating leukocytes, natural killer cells and immunoglobulin concentration increased significantly, indicating that a pre-existing deficiency had been present. The implication is that the deficiency had been due to prolonged exposure to anaesthetic agents. Moreover, several studies have demonstrated the ability of anaesthetic agents to alter neutrophil immune function, including chemotactic migration [5-8], microbicidal activity [9-11] and superoxide production [12,13].

Neutrophils are an integral component of the early cell-mediated immune response and are the principal cells activated in the initial response to surgical trauma. Rapid systemic neutrophilia accompanies such insults and restoration of homeostasis and termination of the immune response are partly achieved via apoptosis, genetically regulated cell suicide [14-18]. Apoptosis plays an important role in regulating the function of neutrophils because it limits their phlogistic potential [19,20]. During inflammation, apoptosis is necessary for removal of extravasated neutrophils from the inflamed site by macrophage phagocytosis [21]. This mechanism of clearance limits release of reactive oxygen species and degradative enzymes and thus inflammation-related tissue injury because release of reactive oxygen species and degradative enzymes contained in the neutrophil is prevented.

Apoptosis may also be influenced by chronic occupational exposure to volatile anaesthetic agents [22]. In comparing neutrophil apoptosis in healthcare workers (n = 20) with unexposed volunteers (n = 10), Goto and colleagues [22] found that the per cent apoptosis was less at 24- (but not at 1- and 12-) h culture in healthcare workers (50.5 (9.7)%; P = 0.008) than in unexposed volunteers (57.3 (5.1)%). Further evidence in support of the ability of volatile anaesthetic agents to affect apoptosis has recently emerged [23] in which it was demonstrated that norepinephrine-induced apoptosis was inhibited in rat myocytes by exposure to volatile anaesthetic agents.

The objective of the present study was to compare the rate of neutrophil apoptosis in unexposed volunteers with that in anaesthetists chronically exposed to volatile anaesthetic agents.


With institutional ethics approval and having obtained written informed consent from each, 12 anaesthetists and 12 volunteers (matched for age, gender and smoking habit) were recruited. Samples were withdrawn from the anaesthetists during the course of their normal working day and processed immediately afterwards. The number of years of each anaesthetist's occupational exposure to volatile anaesthetic agents was documented. Exclusion criteria were surgery within the past 6 months, concurrent medication and recent (1 month) infection.

Preparation of purified populations of neutrophils

Neutrophils were isolated by sequential sedimentation in 6% Dextran® (520 000 Da; Sigma, Poole, Dorset, UK) in 0.9% sodium chloride for 45 min at 22°C, centrifugation in Ficoll-Paque® (Pharmacia LKB Biotechnology, Piscataway, NJ, USA) at 300 g for 30 min to pellet granulocytes and the remaining erythrocytes, and centrifugation of the resuspended pellet over an 81% isotonic Percoll® (Sigma) gradient at 350 g for 15 min to pellet erythrocytes. The diffuse layer at the interface containing neutrophils was then harvested, washed, resuspended in medium and counted. Cell viability was assessed using tryphan blue exclusion. The proportion of neutrophils in the preparation was determined using Rapi-diff II® (Diagnostic Developments, Skelmersdale, Lancs, UK) staining on cytocentrifuged samples.

Culture conditions for neutrophils

Preparations of isolated neutrophils were maintained in RPMI 1640 supplemented with 10% autologous PPP, and 2 mmol L-glutamine, 100 U mL−1 penicillin, 100 μg mL−1 streptomycin and 2.5 μg mL−1 amphotericin B at a concentration of 106 cells mL−1 in ultralow attachment bottle at 37°C in a humidified CO2 incubator (5% CO2-95% air).

Immunofluorescence flow cytometry of annexin V-FITC binding

Translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane is an early event in apoptosis, occurring before any nuclear changes had occurred. The binding of annexin V-FITC® (Bender MedSystems, Vienna, Austria) to phosphatidylserine in a Ca2+-dependent manner was used as a sensitive measure of neutrophil apoptosis. Briefly, neutrophils (0.5 × 106 cells mL−1) were dualstained with propidium iodide (Sigma) (final concentration 10 μg mL−1) and annexin V-FITC (final concentration 0.6 μg mL−1) diluted in binding buffer (10 mmol Hepes/NaOH, pH 7.4; 140 mmol NaCl; 2.5 mmol CaCl2) for 5 min at room temperature. Neutrophils were analysed using a Becton Dickinson FACScan® flow cytometer (Becton Dickinson, Cowley, Oxford, UK) equipped with CellQuest® software with excitation at 488 nm and emission collected through 530/30 band-pass filter for FITC (FL1-H) and a 585/42 band-pass filter for propidium iodide (FL2-H). Ten thousand events were collected while gating on physical parameters to exclude cell debris.

Data were analysed using paired, one-tailed t-tests. P < 0.05 was considered as significant.


Twelve anaesthetists (aged 48.2 (7.9) range 39-60 yr) and 12 unexposed volunteers (48.4 (9.3) range 38-63 yr) matched for age [4], gender and smoking habit [24] were recruited (Table 1). All but one participant in each group were non-smokers. The anaesthetist who smoked and his match reported three- and five-pack per year smoking histories respectively.

Table 1
Table 1:
Exposed anaesthetists matched with unexposed volunteers for gender, age, weight, and smoking habit.

Figure 1 compares the rate of neutrophil apoptosis in exposed anaesthetists and unexposed volunteers at 1, 12 and 24 h. All data are mean (SD). Data are reported as the percentage of annexin V-FITC-positive cells with an intact cell membrane, i.e. the proportion of apoptotic cells in the sample analysed. At 1 h in culture (but not at 12 or 24 h), the rate of neutrophil apoptosis was significantly less in anaesthetists (13.8 (12.9 SD)%) in comparison with the unexposed volunteers (34.4 (12.1 SD)%; P = 0.001). No significant correlation was demonstrable between the years of potential exposure and apoptotic rate (r2 = −0.58 (1 h) (Fig. 2), 0.13 (12 h), and 0.06 (24 h)).

Figure 1
Figure 1:
Percentage neutrophil apoptosis in healthcare workers and unexposed volunteers at 1, 12 and 24 h in culture. *P < 0.05; •: exposed; ○: unexposed.
Figure 2
Figure 2:
Correlation between anaesthetists' years of exposure to inhalational anaesthetics and rate of neutrophil apoptosis at 1 h in culture. Data are the percentage of annexin V-FITC-positive cells with an intact cell membrane reflecting the relative proportion of apoptotic cells. Data are the mean (SD).


The most important finding was the demonstration of a significant (P < 0.05) decrease in neutrophil apoptosis at 1 h in culture in anaesthetists who have been occupationally exposed to volatile anaesthetic agents with unexposed volunteers. This observation is consistent with previous in vivo experiments performed at our institution (N. F. Fanning, personal communication), which demonstrated a significant (P = 0.05) inhibition of neutrophil apoptosis at 1, 12 and 24 h in culture following exposure to anaesthetic concentrations of isoflurane (2%) and propofol (0.6 μg mL−1). The data are also consistent with those of a previous occupational exposure study performed at our institution [22] that examined the effects of acute exposure to anaesthetics and in which the per cent apoptosis was less at 24-(but not at 1- and 12-) h culture in anaesthetists (50.5 (9.7)%; P = 0.008) than in unexposed volunteers (57.3 (5.1)%). We believe that a 2.5-fold difference in neutrophil apoptosis at 1 h may have real clinical implications, particularly given that the effect was demonstrated in a working environment where exposure concentrations were below the recommended limits. Although we cannot satisfactorily explain why inhibition is demonstrated at 1 but not at 12 and 24 h, it is probably due to the small group number and considerable intersubject variation.

Occupational exposure standards were introduced for nitrous oxide (100 ppm), halothane (10 ppm), isoflurane (50 ppm) and enflurane (50 ppm) in January 1996 as part of the Control of Substances Hazardous to Health Regulations (Health Services Advisory Committee. Anaesthetic Agents: Controlling Exposure under COSHH. London, UK: HMSO, 1995). However, occupational exposure standards for sevoflurane and desflurane have yet to be established. According to the US Occupational Health and Safety Administration, no worker should be exposed to concentrations of waste anaesthetic gases > 2 parts per million (ppm) of any halogenated anaesthetic agent, based on the weight of the agent collected for a 45 L air sample by charcoal adsorption over a sampling period not exceeding 1 h.

Goto and colleagues [22], at our institution, established that the hospital's isoflurane (scavenged = 0.2 ± 0.3 ppm; unscavenged = 0.3 ± 0.2 ppm) and nitrous oxide (scavenged = 39.5 ± 37.2; unscavenged = 26.0 ± 16.1 ppm) exposure concentrations were below the recommended exposure standards. Exposure concentrations for sevoflurane were 1.1 ± 0.7 ppm in the scavenged theatres and 0.8 ± 1.5 ppm in the unscavenged theatres, but, as yet, no recommended occupational exposure limit exists for sevoflurane.

The strengths of the present study include the fact that the participating anaesthetists were matched for gender, age [4], and smoking habit [24] with similar individuals who had never been exposed to anaesthetic agents. Moreover, the potential contribution of many factors that may have otherwise affected the demonstrated inhibition was diminished by the chosen exclusion criteria. The participating anaesthetists were selected mainly from the 40-50 yr age group to increase the likelihood of prolonged environmental exposure. Furthermore, the recruited anaesthetists had all worked during a time when effective theatre scavenging was not universally in place.

These findings must be interpreted in light of the small number of subjects studied and the fact that the anaesthetists are presently working in scavenged theatres in which the environmental concentration of anaesthetic agents is low. It is possible that the effect observed may be due to environmental factors common to operating theatre personnel (such as stress) other than volatile anaesthetic agents. The absence of a significant correlation may be due to the crude means (years at work) used to estimate the degree of cumulative exposure.

Many studies have outlined the potential hazards arising from chronic exposure to anaesthetic agents. Rowland and colleagues [1] demonstrated that decreased fertility among females employed as dental assistants was due to high levels of exposure to nitrous oxide. In a retrospective study of personnel exposed to anaesthetic gases in operating and recovery rooms in Ontario hospitals, Guirguis and colleagues [2] showed that females exposed to such gases had significantly increased frequencies of spontaneous abortion and their children had significantly more congenital abnormalities (P < 0.05). A similar investigation by Vainio [3] indicated that there is an excess risk of spontaneous abortions among operating room staff and that the causative agent is nitrous oxide. In addition to being linked to carcinogenic and teratogenic complications, Peric and colleagues [25] posited that waste anaesthetic gases caused immunological disturbances in anaesthetic personnel. Apoptosis may contribute to the aforementioned conditions through its central involvement in such diverse processes as embryological remodelling, haematopoesis, immune tolerance and inflammation [26-28].

These findings provide further evidence that a moderate degree of inhibition of neutrophil apoptosis occurs in those chronically exposed to anaesthetic agents. Given that neutrophil apoptosis is a crucial step in terminating the inflammatory response, it is speculated that dysregulation of this process could result in augmentation of the injury of inflamed tissues. It is also possible that pharmacological inhibition of one cell death pathway (i.e. apoptosis) will divert neutrophils towards the alternative necrotic pathway, thus increasing the degree of tissue injury. Critically, the functional state of neutrophils in which apoptosis has been pharmacologically inhibited has not been defined. The implications for healthcare workers in terms of defence against infection and nature of inflammatory response warrant further investigation.


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ANAESTHESIA; CELL DEATH, apoptosis; PATHOLOGICAL PROCESSES, inflammation; IMMUNE SYSTEM, leukocytes, granulocytes, neutrophils; ENVIRONMENTAL POLLUTION, environmental exposure, occupational exposure

© 2002 European Society of Anaesthesiology